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VanNieuwenhze, M. S.

Publications and source records attributed to VanNieuwenhze, M. S..

7 recordsLinked to original sources

Phenotypic plasticity in bacterial elongation among closely related species

Cell elongation is a fundamental component of the bacterial cell cycle and has been studied over many decades, in part owing to its mechanisms being a target of numerous antibiotic classes. While several distinct modes of cell elongation have been described, these studies have largely relied on a handful of model bacterial species. Therefore, we have a limited view of the diversity of cell elongation approaches that are employed by bacteria, and how these vary in response to evolutionary and environmental influences. Here, by employing fluorescent D-amino acids (FDAAs) to track the spatiotemporal dynamics of elongation, we reveal previously unsuspected diversity of elongation modes among closely related species of the Caulobacteraceae, with species-specific combinations of dispersed, midcell and polar elongation that can be either unidirectional or bidirectional. Using genetic, cell biology, and phylogenetic approaches, we demonstrate that evolution of unidirectional-midcell elongation is accompanied by changes in the localization pattern of the peptidoglycan synthase PBP2 and infer that elongation complexes display a high degree of phenotypic plasticity, both among the Caulobacteraceae and more widely among the Alphaproteobacteria. Demonstration that even closely related bacterial species employ highly distinct mechanisms of cell elongation reshapes our understanding of the evolution and regulation of bacterial cell growth, with broad implications for bacterial morphology, adaptation, and antibiotic resistance.

microbiology↗

Unipolar peptidoglycan synthesis in the Rhizobiales requires an essential class A penicillin-binding protein

Members of the Rhizobiales are polarly-growing bacteria that lack homologs of the canonical rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant-pathogen Agrobacterium tumefaciens. The development of fluorescent O_SCPLOWDC_SCPLOW-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis convincingly demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an alternative mode of cell wall synthesis that likely requires O_SCPLOWLDC_SCPLOW-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation.

microbiology↗

Organization of Peptidoglycan Synthesis in Nodes and Separate Rings at Different Stages of Cell Division of Streptococcus pneumoniae

Bacterial peptidoglycan (PG) synthesis requires strict spatial and temporal organization to reproduce specific cell shapes. In the ovoid-shaped, pathogenic bacterium Streptococcus pneumoniae (Spn), septal and peripheral (sidewall-like) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Using fluorescent D-amino acid (FDAA) probes, we show that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Short pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Control experiments in wild-type and mutant strains support the interpretation of nodal spacing of TP activity, and statistical analysis confirmed that the number of nodes correlates with different ring diameters. This nodal pattern of FDAA labeling is conserved in other ovoid-shaped species. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal a highly ordered PG synthesis apparatus in ovococcal bacteria at different stages of division. SIGNIFICANCEThe spatial organization of PBPs and their TP activity at division septa is not well understood. In some bacteria, TP activity and PBP localization seem to be nodal (also called punctate), whereas in other bacteria, discrete foci of PBP activity are infrequently or not observed. Here we report two basic properties of the organization of PBPs and TP activity in the ovoid-shaped bacterium Spn. First, there is distinct spatial separation of the sPG machine, including FtsZ, from the pPG synthesis machine at the midcell of dividing Spn cells. Second, in predivisional cells, PBPs and TP activity are organized heterogeneously into regularly spaced nodes, whose number and dynamic distribution are likely driven by the PG synthesis of PBP:SEDS complexes.

microbiology↗

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth and structural integrity. PG is synthesized by two different types of synthase complexes (class A Penicillin-binding Proteins [PBP]s/Lpos and Shape, Elongation, Division, Sporulation [SEDS]/class B PBP pairs) and degraded by autolytic enzymes to accommodate growth processes. It is thought that autolsyin activity (and particulary the activity of endopeptidases, EPs) is required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and the separate synthesis machineries remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological defects, increased cell mass, a decline in viability, and continuing (yet aberrant) incorporation of cell wall material. Mass increase and cell wall incorporation proceeded in the presence of Rod system inhibitors, but was abolished upon inhibition of aPBPs. However, the Rod system remained functional (i.e., exhibited sustained directed motion) even after prolonged EP depletion, without effectively promoting cell elongation. Lastly, heterologous expression of an EP from Neisseria gonorrhoeae could fully complement growth and morphology of an EP-insufficient V. cholerae. Overall, our findings suggest that in V. cholerae, the Rod system requires endopeptidase activity (but not necessarily direct interaction with EPs) to promote cell expansion and substantial PG incorporation, whereas aPBPs are able to engage in sacculus construction even during severe EP insufficiency. ImportanceSynthesis and turnover of the bacterial cell wall must be tightly co-ordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are required only for substantial PG incorporation mediated by the Rod system, while the aPBPs maintain structural integrity during endopeptidase insufficiency. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the co-ordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

microbiology↗

A Division of Labor in the Recruitment and TopologicalOrganization of a Bacterial Morphogenic Complex

Bacteria come in an array of shapes and sizes, but the mechanisms underlying diverse morphologies are poorly understood. The peptidoglycan (PG) cell wall is the primary determinant of cell shape. At the molecular level, morphological variation often results from the regulation of enzymes involved in cell elongation and division. These enzymes are spatially controlled by cytoskeletal scaffolding proteins, that both recruit and organize the PG synthesis complex. How then do cells define alternative morphogenic processes that are distinct from cell elongation and division? To address this, we have turned to the specific morphotype of Alphaproteobacterial stalks. Stalk synthesis is a specialized form of zonal growth, which requires PG synthesis in a spatially constrained zone to extend a thin cylindrical projection of the cell envelope. The morphogen SpmX defines the site of stalk PG synthesis, but SpmX is a PG hydrolase. How then does a non-cytoskeletal protein, SpmX, define and constrain PG synthesis to form stalks? Here we report that SpmX and the bactofilin BacA act in concert to regulate stalk synthesis in Asticcacaulis biprosthecum. We show that SpmX recruits BacA to the site of stalk synthesis. BacA then serves as a stalk-specific topological organizer for PG synthesis activity, including its recruiter SpmX, at the base of the stalk. In the absence of BacA, cells produce "pseudostalks" that are the result of unconstrained PG synthesis. Therefore, the protein responsible for recruitment of a morphogenic PG remodeling complex, SpmX, is distinct from the protein that topologically organizes the complex, BacA.

microbiology↗

SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely through the localisation of the late stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and the bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in cell elongation and cell division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 likely constitute cognate pairs of interacting proteins. Lack of RodA-PBP3 decreased cell eccentricity due to deficient pre-septal PGN synthesis, whereas the depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilising the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings/arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate lateral and septal PGN incorporation, respectively, and that the activity of these pairs must be balanced in order to maintain coccoid morphology.

microbiology↗

Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori

Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. We show that the helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod bacteria. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.

microbiology↗